Cnpy1 within DFCs regulates DFC clustering. (A and B) dp-Erk staining in DFC^control-MO–injected (A) or DFC^cnpy1-MO–injected (B) Tg[sox17:GFP] embryos at 60% epiboly stage. (Scale bar: 20 μm.) dp-Erk signals (red) were down-regulated in GFP-positive DFCs (green). (C and D) cnpy1 (purple) and sox32 (red) expression in DMSO-treated (C) or SU5402-treated (D) embryos at 60% epiboly stage. (Scale bar: 200 μm.) Dotted lines in A–D mark the outlines of DFC populations. (E–J) sox32 expression in DFC^control-MO (E), DFC^cnpy1-MO (F), yolk^control-MO (G), yolk^cnpy1-MO (H), DFC^{cnpy1-MO+mRFP} (I), or DFC^{cnpy1-MO+Cnpy1} (J) embryos at 70% epiboly stage. Dorsal view, anterior to the top. (Scale bar: 200 μm.) (E2–J2) Higher-magnification images highlight DFCs. (K) Percentages of normal (clustered) or broken-up DFCs were scored by using the sox32 expression pattern in uninjected (n = 68), cnpy1-MO (n = 77), DFC^control-MO (n = 61), DFC^cnpy1-MO (n = 78), yolk^control-MO (n = 56), yolk^cnpy1-MO (n = 62), DFC^{cnpy1-MO+mRFP} (n = 119), or DFC^{cnpy1-MO+Cnpy1} (n = 123) embryos. Statistically significant (P < 0.05) differences could be seen in uninjected versus cnpy1-MO, DFC^control-MO versus DFC^cnpy1-MO, and DFC^{cnpy1-MO+mRFP} versus DFC^{cnpy1-MO+Cnpy1} embryos.

Cnpy1 function within DFCs is essential for ciliogenesis and LR patterning. (A and B) A-tubulin (green) and nucleus (red) staining in uninjected (A) or DFC^cnpy1-MO–injected (B) embryos at the six-somite stage. Vegetal pole view. (Scale bars: 20 μm.) (A2 and B2) X–Z view around the KV. Lumen formation was not completed in DFC^cnpy1-MO–injected embryos (B2). (C) Number (red) or length (blue) of KV primary cilia in uninjected (n = 10 or 49), DFC^cnpy1-MO (n = 10 or 48), yolk^control-MO (n = 11 or 77), yolk^cnpy1-MO (n = 11 or 58), DFC^{cnpy1-MO+mRFP} (n = 10 or 61), or DFC^{cnpy1-MO+Cnpy1} (n = 11 or 85) embryos. (Error bars show SEM.) Statistically significant (P < 0.05) differences could be seen in uninjected versus DFC^cnpy1-MO and DFC^{cnpy1-MO+mRFP} versus DFC^{cnpy1-MO+Cnpy1} embryos. (D and E) Representative images of mlc2a expression demonstrating normal looping (uninjected; D) or reversed looping (cnpy1-MO; E) of the heart in embryos at the high pec stage. Ventral view, anterior to the top. A, atrium; V, ventricle. (F) Percentages of normal looping, reversed looping, no looping, or cardia bifida of the heart in uninjected (n = 164), control-MO (n = 118), cnpy1-MO (n = 119), fgf8-MO (n = 65), DFC^control-MO (n = 95), DFC^cnpy1-MO (n = 146), DFC^cdh1-MO (n = 106), yolk^control-MO (n = 96), yolk^cnpy1-MO (n = 94), DFC^{cnpy1-MO+mRFP} (n = 136), and DFC^{cnpy1-MO+Cnpy1} (n = 165) embryos. Statistically significant (P < 0.05) differences could be seen in uninjected versus cnpy1-MO, DFC^control-MO versus DFC^cnpy1-MO, and DFC^{cnpy1-MO+mRFP} versus DFC^{cnpy1-MO+Cnpy1} embryos.

FGF signaling plays crucial roles in DFC clustering and KV ciliogenesis. (A and B) sox32 (A) or no tail (B) expression in fgf8-MO–injected embryos. Dorsal view, anterior to the top. (Scale bar: 200 μm.) (A2 and B2) Higher-magnification images highlight DFCs. (B2, D, and E) The white dotted lines mark the boundary between DFCs and the blastoderm margin. (C) Percentages of normal or broken-up DFCs were scored by using the sox32 or no tail expression patterns in uninjected (n = 68 or 89) or fgf8-MO (n = 61 or 69) embryos. Statistically significant (P < 0.05) differences could be seen in uninjected versus fgf8-MO embryos. (D–K) Transient activation of FGF signaling restored the broken-up DFC phenotype (D–F), ciliogenesis (G–J), and cardiac laterality (K) in DFC^cnpy1-MO embryos. (D and E) Expression of no tail in DFC^{cnpy1-MO+iFGFR1} embryos treated with ethanol (D) or AP20187 (E). (F) Percentages of broken-up DFC phenotype in ethanol-treated (n = 84) or AP20187-treated (n = 93) DFC^{cnpy1-MO+iFGFR1} embryos. The conditional activation of Fgfr1 after treatment with AP20187 significantly decreased the broken-up DFC phenotype (67%; P < 0.05) (G–J) A-tubulin (green) staining in ethanol-treated (G) or AP20187-treated (H) DFC^{cnpy1-MO+iFGFR1} embryos at the six-somite stage. (Scale bar: 20 µm.) (I and J) Number (I) or length (J) of KV primary cilia in ethanol-treated DFC^{cnpy1-MO+iFGFR1} (n = 9 or 36) or AP20187-treated DFC^{cnpy1-MO+iFGFR1} (n = 8 or 34) embryos at the six-somite stage. (Error bars show SEM.) Statistically significant (P < 0.05) differences could be seen in ethanol-treated versus AP20187-treated DFC^{cnpy1-MO+iFGFR1} embryos. (K) Percentages of cardiac laterality defect in ethanol-treated (n = 89) or AP20187-treated (n = 102) DFC^{cnpy1-MO+iFGFR1} embryos. The conditional activation of Fgfr1 after treatment with AP20187 alleviated the cardiac laterality defect (48%; P < 0.05).

cdh1 expression. (A and B) cdh1 (purple) and sox32 (red) expression in DFC^control-MO (A) or DFC^cnpy1-MO (B) embryos at 65% epiboly stage. (C and D) tbx16 (purple) and sox32 (red) expression in DFC^control-MO (C) or DFC^cnpy1-MO (D) embryos at 65% epiboly stage. Dotted lines in A–D mark the outlines of DFC populations. (Scale bar: 200 μm.) (E and F) Expression of sox32 in DFC^{cnpy1-MO+mRFP} (E) or DFC^{cnpy1-MO + Cdh1} (F) embryos at 80% epiboly. (Scale bar: 200 μm.) (G) Percentage of broken-up DFC phenotype in mRFP-overexpressing (n = 82) or Cdh1-overexpressing (n = 103) DFC^cnpy1-MO embryos. Overexpression of Cdh1 rescued the broken-up DFC phenotype in DFC^cnpy1-MO embryos (60%; P < 0.05). (H) Diagram illustrating the FGF-dependent cell–cell communication control mechanisms of the forerunner cell cluster during early development. The model depicts the activation of intracellular FGF signaling via binding of Fgf8 ligands and Fgfr1 on the cell surface of two adjacent DFCs (blue ovals). The amplified FGF signal, through Cnpy1-mediated maturation of Fgfr1 within DFCs, subsequently activates the expression of tbx16 and cdh1 to organize forerunner cells as a cluster.

Expression of cnpy1 in zebrafish early embryos. (A, B) Expression of cnpy1 in embryos at 80% epiboly (A), or at the 6-somite stage (B). (A) Dorsal view, anterior to the top. (A′) Higher-magnification image highlights DFCs. The white dotted line marks the boundary between DFCs and the blastoderm margin. (B) Lateral view, anterior to the left. (B′) Flat-mounted embryo at the 6-somite stage. Dorsal view, anterior to the left. cnpy1 expression at the 6-somite stage is restricted to the polster, MHB and tailbud.

A positive control loop between Cnpy1 and FGF signaling is established in DFCs. (A-C) dp-Erk staining in uninjected (A), cnpy1-MO-injected (B) or DFC^cnpy1-MO-injected (C) embryos at 60% epiboly stage. Scale bar: 200 μm. (D, E) dp-Erk staining in control-MO-injected (D) or cnpy1-MO-injected (E) Tg[sox17:GFP] embryos at 60% epiboly stage. Scale bar: 20 μm. dp-Erk signals (red) were down-regulated in GFP-positive DFCs (green). (F, G) cnpy1 (purple) and GFP (red) expression in control-MO-injected (F) or fgf8-MO-injected (G) Tg[sox17:GFP] embryos at 60% epiboly stage. Scale bar: 200 μm. Dotted lines mark the outlines of DFC populations.

cnpy1 function is essential for DFC clustering. (A, B) sox32 expression in uninjected (A) or cnpy1-MO-injected (B) embryos. Scale bar: 200 μm. (C) Percentages of normal or broken-up DFCs were scored using the no tail expression pattern in uninjected (n = 84), cnpy1-MO (n = 55), DFC^control-MO (n = 64), DFC^cnpy1-MO (n = 48), yolk^control-MO (n = 50) or yolk^cnpy1-MO (n = 66) embryos. Statistically significant (P < 0.05) differences could be seen in uninjected versus cnpy1-MO (P = 2.76 x 10^-9) and DFC^control-MO versus DFC^cnpy1-MO (P = 6.1 x 10^-5), while no difference was seen in uninjected versus DFC^control-MO (P = 0.403), uninjected versus yolk^control-MO (P = 0.361), DFC^control-MO versus yolk^control-MO (P = 1.000) or yolkcontrol-MO versus yolkcnpy1-MO (P = 0.314). (D-G) no tail expression in uninjected (D), cnpy1-MO-injected (E), DFC^control-MO (F) or DFC^cnpy1-MO (G) embryos. Dorsal view, anterior to the top. Scale bar: 200 μm. (A′-D′) Higher-magnification images highlight DFCs. The white dotted lines mark the boundary between DFCs and the blastoderm margin.

DFC-specific knockdown of cnpy1 does not affect DFC migration towards the vegetal pole. (A-J) Time-lapse confocal imaging of DFC migration in DFC^control-MO-injected (A-E) or DFC^cnpy1-MO-injected (F-J) embryos. DFCs were labeled with SYTO17 tracer, and DFC migration was monitored every 2.5 min for 82.5 min. A, F; 0 min, B, G; 20 min, C, H; 40 min, D, I; 60 min. Although a DFC cluster (arrow in A) is found in the DFC^control-MO embryo at 0 min, sparse DFC populations (arrows in F) appear in the DFC^cnpy1-MO embryo. AP, animal pole; VP, vegetal pole. (E, J) Three cells in each embryo at 0 min are marked by red dots, and their migration is traced at 20-min intervals (indicated by color changes from red [0 min] to green [60 min]).

The broken-up DFC phenotype in DFCcnpy1-MO embryos may interfere with proper recruitment of DFCs to the KV. (A-D) DFC morphology in Tg[sox17:GFP] embryos injected with control-MO (A, C; DFC^control-MO) or cnpy1-MO (B, D; DFC^cnpy1-MO). (A, B) Dorsal view of the embryos at 60% epiboly stage. (C, D) Vegetal pole view of the embryos at bud stage. Scale bar: 20 μm. (E) Number of DFCs scored by GFP expression.

Loss of FGF signaling leads to defects in KV formation and LR patterning. (A, B) Representative images showing horseshoe-shaped (uninjected; A) or abnormal (DFC ^cnpy1-MO; B) patterns of charon expression in embryos at the 6-somite stage. Vegetal pole view. Scale bar: 200 μm. (C) Percentages of normal or abnormal phenotypes were scored using the charon expression pattern in uninjected (n = 54), cnpy1-MO (n = 73), fgf8-MO (n = 66), DFC^cnpy1-MO (n = 72) or DFC^cdh1-MO (n = 71) embryos. Statistically significant (P < 0.05) differences could be seen in uninjected versus cnpy1-MO (P = 5.66 x 10^-8), fgf8-MO (P = 4.99 x 10^-7), DFC^cnpy1-MO (P = 4.14 x 10^-7) and DFC^cdh1-MO (P = 6.40 x 10^-8). (D, E) Representative images demonstrating left-sided (uninjected; D) or bilateral (cnpy1-MO; E) expression of spaw at the 20-somite stege. Dorsal view, anterior to the top. Scale bar: 200 μm. (F) Percentage of left-sided, right-sided, bilateral, or no (absent) expression of spaw in uninjected (n = 156), cnpy1-MO (n = 133), fgf8-MO (n = 108), DFC^cnpy1-MO (n = 110) or DFC^cdh1-MO (n = 84) embryos. Statistically significant (P < 0.05) differences could be seen in uninjected versus cnpy1-MO (P < 2.2 x 10-16), fgf8-MO (P = 4.96 x 10-16), DFC^cnpy1-MO (P < 2.2 x 10^-16) and DFC^cdh1-MO (P = 9.21 x 10^-11).

Cnpy1 function in DFCs is required for ciliogenesis in the KV. (A-D) A-tubulin staining in yolk^control-MO (A), yolk^cnpy1-MO (B), DFC^{cnpy1-MO+mRFP} (C) and DFC^{cnpy1-MO+Cnpy1} (D) embryos at the 6-somite stage. Vegetal pole view. Scale bar: 20 μm.

ace/fgf8 mutants result in the broken-up DFC phenotype. (A-C) Upper panels indicate sox32 expression in wild type (+/+; A), ace heterozygote (ace/+; B), ace homozygote (ace/ace; C) at 70% epiboly. Scale bar: 200 μm. Lower panels show sequences around the ace mutation. Arrows indicate the position of the ace mutation. Substitution from G to A occurs in the ace allele. (D) Percentages of normal or broken-up DFCs were scored using the sox32 expression pattern in wild type (n = 17), ace heterozygote (n = 36) or ace homozygote (n = 14).

DFC-specific knockdown of cnpy1 attenuates F-actin accumulation at the cell-cell contact sites of DFCs. (A, B) Distribution of F-actin (red) and FITC-labeled MO (green) in DFC^control-MO (A) or DFC^cnpy1-MO (B) embryos. Dorsal view, anterior to the top. Scale bar: 20 μm. White dotted lines mark the plasma membrane outlines of MO-containing DFC populations. (A’, B’) Higher-magnification images highlight MO-containing DFCs. In control-MO-containing DFCs (A’), F-actin accumulated at the cell-cell contact sites of the plasma membrane. However, accumulation of F-actin was limited in cnpy1-MO-containing DFCs (B’).

A genetic cascade including tbx16 and cdh1 mediates FGF signaling in DFCs. (A, B) Dorsal view of tbx16 expression in uninjected (A) or DFC^cnpy1-MO (B) embryos at 65% epiboly stage. Scale bar: 200 μm. (C-E) Dorsal view of cdh1 expression in uninjected (C), DFC^cnpy1-MO-injected (D) or DFCtbx16-MO-injected (E) embryos at 65% epiboly stage. Scale bar: 200 μm. (A′-E′) Higher-magnification images highlight DFCs. (F, G) cdh1 (purple) and GFP (red) expression in DFC^control-MO-injected (F) or DFC^tbx16-MO-injected (G) Tg[sox17:GFP] embryos at 60% epiboly stage. Scale bar: 200 μm. Dotted lines in panels A′-E′, F and G mark the outlines of the DFC populations.

DFC-specific knockdown of tbx16 or cdh1 results in the broken-up DFC phenotype. (A-C) Dorsal view of sox32 expression in uninjected (A), DFC^tbx16-MO (B) or DFC^chd1-MO (C) embryos at 65% epiboly stage. Dorsal view, anterior to the top. Scale bar: 200 μm. (D) Percentages of normal or broken-up DFCs were scored using the sox32 expression pattern in uninjected (n = 64), DFC^tbx16-MO (n = 55), DFC^cdh1-MO (n = 69) yolk^tbx16-MO (n = 61) and yolk^cdh1-MO (n = 65) embryos. Statistically significant (P < 0.05) differences could be seen in uninjected versus DFC^tbx16-MO (P = 1.68 x 10^-4) and DFC^cdh1-MO (P = 1.17 x 10^-8), but not between uninjected and yolk^tbx16-MO (P = 1.00) or yolk^cdh1-MO (P = 1.00).

DFC-specific overexpression of dn-Fgfr1 can lead to broken-up DFC clusters. (A, B) Dorsal view of sox32 expression of DFC^LacZ-injected (A) or DFC^dn-Fgfr1-injected (B) embryos. Scale bar: 200 μm. (C) Percentages of normal or broken-up DFCs were scored using the sox32 expression pattern in DFC^LacZ-injected (n = 54) or DFC^dn-Fgfr1-injected (n = 47) embryos. A significant difference (P = 0.0025) could be seen between DFC^LacZ and DFC^dn-Fgfr1.